This proposal will continue to elucidate the regulation of neuronal function by calcium-binding proteins (CBPs) including calmodulin (CaM)-dependent second messenger systems. Calcium (Ca2+), one of the most important cellular signals, can control events with timings as diverse as neurotransmitter release and neuronal degeneration. Yet, the dynamics of its interactions with CBPs are little known, least of all at the time-scale needed to resolve its distinct roles in neuronal signaling The proposed studies will provide detailed insights into the functioning of endogenous neuronal Ca2+ -buffers, their interactions and competition for Ca2+ with Ca2+ effector systems, and their combined roles in determining the specific vulnerability of central neurons. The main hypothesis is as follows: the roles of neuronal CBPs as intracellular Ca2+ buffers or as competitors for specific Ca2+-dependent regulatory events are defined by their unique Ca2+-binding kinetics. In turn, the distinct kinetics and selective interactions of CBPs with Ca2+-effector systems are responsible for their diverse roles in regulating neuronal excitability, synaptic integration, and selective neuronal vulnerability. This hypothesis will be addressed in the following specific aims: 1) to resolve the Ca2+-binding kinetics of three neuronal CBPs including calbindin-D28K (CB28K) Parvalbumin (PV), and calretinin (CR) at or near physiological conditions; 2) to determine the physiological Ca2+-binding kinetics of substrate-free CaM, and of CaM bound to some effectors including the Ca2+/CAM-dependent protein kinase II (CaMK II), the Ca2+/CaM-dependent serine (Ser)/threonine (Thr) phosphatase calcineurin (CN), and the CaM-binding domain of the SK2 CA2+ dependent potassium (K+) channel; 3) to ascertain the role of CB28K in neuronal Ca2+ handling and in short-term plasticity at specific synapses; 4) to determine how CR alters neuronal excitability and short-term plasticity at synapses between mossy cells and granule cells of the hippocampal formation; 5) to uncover the relationship between the vulnerability of murine mossy cells and their CR content. These five aims will be accomplished by measuring Ca2+-binding to CBPs after ultra-fast photolysis of caged Ca2+ followed by computing the binding rate constants through compartmental kinetic modeling. High-resolution electrophysiological recordings combined with measurements of intracellular Ca2+ dynamics at the sub-millisecond time-scale will be done in identified neurons of brain slices prepared from mice with genetically altered levels of CBPs. For the first time, the physiological Ca2+-binding properties of CBPs will be linked to their functions as Ca2+ buffers, regulators of cellular excitability, and determinants of selective neuronal vulnerability. Understanding the precise relationship between intracellular Ca+ -binding and Ca2+ effector mechanisms will uncover the complex involvement of Ca2+ in the triad of neuronal excitability, plasticity, and vulnerability. This triad is critical in severe neurological maladies including ischemia/stroke neurodegenerative disorders, and epilepsy.